The goal of this research program is to develop a genome-scale in silico model of metabolism, its regulation, and macromolecular synthesis in Saccharomyces cerevisiae using existing genetic/biochemical literature and data as well as experimental data generated within the program. The model will also be used to drive prospective experimental designs, and these experiments will be performed to interrogate the model. Genomics and various high-throughput technologies are generating large volumes of molecular data on living cells. This data and the complexity of cellular processes are now demanding the construction of large-scale (ultimately genome-scale) in silico models in order to interpret the data in the context of the full cellular function of an organism. The proposed research program is comprised of three specific aims: #1 to build a genome-scale model of yeast that describes metabolism and its regulation along with the protein synthesis process based on data available on the individual cellular components; #2 to generate high-throughput functional genomics data to expand and validate the model in an on-going fashion; and #3 to perform modeldriven targeted phenotyping of knock-out strains to validate or refute the model. The data types that will be used to reconstruct the network in Aim #2 are genome-wide transcription profiling and DNA-binding site identification (location analysis) data. These experiments will be designed in a model-driven fashion to allow efficient validation and extension of the existing model. Finally, the model will be validated by performing both high-throughput and detailed phenotyping experiments designed to optimally probe the model for inconsistencies or inaccuracies. S. cerevisiae is an important eukaryotic model organism for basic medical research and of great industrial significance. A genome-scale model of this organism is expected to have wide impact on the development of systems biology and be of broad practical use. The proposed program would result in the development of the first genome-scale integrated model of a eukar-yotic organism and would serve as a stepping stone for building similar models of higher eukaryotes including mouse and human.